Rapid lysis of gram-positive cocci for pulsed-field gel electrophoresis using achromopeptidase.
To adapt pulsed-field gel electrophoresis (PFGE) to a busy clinical laboratory, we evaluated the use of achromopeptidase (ACP) as the sole agent for lysis of a variety of gram-positive cocci. Growth in an appropriate broth media was adjusted to a 3.0 McFarland density before aliquotting for washing and plug preparation. ACP incorporated into the agarose plugs, or added to the buffer solution, lysed cells in < 1 h when suspended in a Tris-NaCl buffer. Two 30-min washes in a Tris-EDTA buffer were sufficient to remove cellular debris without additional deproteination. Final PFGE results were obtained in approximately 48 h, including the initial broth subculturing.
Leonard
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RB
Associated Regional and University Pathologists, Inc., Salt Lake City, Utah, USA.
Carroll
K C
KC
D015373:Bacterial Typing Techniques; D001433:Bacteriolysis / Q000187:drug effects*; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D016983:Enterococcus / Q000502:physiology; D005843:Geography; D006095:Gram-Positive Cocci / Q000502:physiology*; D012697:Serine Endopeptidases / Q000494:pharmacology*; D013211:Staphylococcus aureus / Q000502:physiology; D013296:Streptococcus pneumoniae / Q000502:physiology; D013297:Streptococcus pyogenes / Q000502:physiology
D012697:Serine Endopeptidases; C036813:lysyl endopeptidase